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Organochemistry of cryoinjury and cryophylaxis 1. Oligomer dissociation as the general mechanism
310
ANNUAL
MEETING
Groups of rats of both sexes were exposed to temperatures of 1°C and -10°C for 60 min prior to orogastric intubation, whereby 2 &i/l00 g body wt was administered. 4Ca level in bone was measured by sbandard monitoring equipment 24 hr after intubation. Exposure to 1°C did not produce significant difference in radiocalcium bone uptake (63 nmCi/g bone wt), when compared to the controls (57 nmCi/g bone wt). In rats exposed to -10°C the mean bone uptake was 92 nmCi/g bone wt, an increase of 61%. A 1-hr exposure to -10°C after intubation also produced an increase (83%) in “Ca uptake (mean 105 nmCi/g bone wt). These results suggest that exposure to low temperature, both prior and after 45Ca administration produces significant increase in calcium bone uptake. Several possible mechanisms should be considered, including hormonal stimulation by hypothermia or increased oxygen consumption and ATP synthesis. 13. Quantitative
Changes in Total Nucleic and Proteins of the Isolated Rabbit During Normothermic and Hypothermic
Acids Heart Per-
fusion. G. I. MALININ, R. M. NARDONE. .4x11 J. (Department of Biology, GeorgeA. PA~SKA
SESSION
ABSTRACTS town University, Washington, DC 20007 and Department of Biology, Catholic University of America, Washington, DC 20011).
Thirty-one rabbit hearts were perfused for 26 hr at normothermic and hypothermic temperatures utilizing a Lindbergh-Rockefeller Institute pulsatile perfusion apparatus. The perfusate consisted of Hanks’ balanced salt solution supplemented with 10% (v/v) rabbit serum. Each atrium and ventricle was then analyzed for total DNA, RNA and protein content utilizing a modified Schmidt-Thannhauser procedure. As compared with controls. levels of DNA and protein were reduced significantly (P < 0.05) in the atria and vent,ricles of the perfused hearts while RNA levels remained unchanged with the exception of the right ventricle where they also depressed. The RNA/DNA ratios, however, were significantly elevated (P < 0.05) in ventricles but remained within normal ranges in atria. The DNA/ protein ratios were depressed in the right ventricles and remained unchanged in the rest of the heart. The RNA/protein ratios were higher in all cardiac chambers. (Supported by Contract N0014-69-A0220 from the Office of Naval Research.)
B : MOLECULAR
14. Fish Blood Glycoproteins with Antifreeze Properties. J. A. RAYMOSD,* AND A. L. DEVRIES
(Physiological Research Laboratory, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92037). The blood of some species of Antarctic fish has been shown to contain freezing point depressing glycoproteins. In a solution having normal colligative properties, the freezing point depression is solely a function of the number of particles in solution. Unlike other solutes in the blood, the glycoproteins are large molecules (2000-20,000 daltons) and have an effect on the freezing point depression two orders of magnitude greater than would be expected on a purely molar basis. This paper is concerned with some of the other ways in which this nonclassical freezing behavior is manifested. Evidence that the glycoproteins are incorporated into the ice phase in a freezing solution is presented. Supercooling of the blood in the presence of an ice seed has also been observed. A possible means by which these phenomena may occur is suggested. (Supported by Grant No. GV 27327 from the National Science Foundation.)
MECHANISMS
WOOD-SMITH, AKD C. WARBY* (Department of Biological Sciences, University of Victoria, Victoria: B.C., Canada).
Experiments are reported that clearly demonstrate, in vitro, the protective action of a number of cryoprotective agents against the inactivat.ion of the enzyme catalase by freezing and thawing. The most significant outcome of these investigations is the very large difference in effectiveness between PVP and either glycerol or dimethyl sulfoxide. The amounts required, on a molar basis, to protect enzyme activity by 50% were approximately 2 X IOF2M (glycerol or DMSO) and 1 X IO-’ III (PVP of av MW 40,000 daltons). This very large difference in effectiveness, nearly 1000 even on a weight basis, is difficult to explain in terms of the slight but definite colligative properties of PVP. Other polymers such as dextran protect catalase but are far less effective than PVP. Possible mechanisms of PVP protection will be discussed. (Supported by Canadian Medical Research Council Grant MA4055.) 16. Organochemistry phylaxis
15. Protective Efiects of Low an,d High Molecular Weight Compounds on the Stability of Catalase Subjected to Freezing and Thawing. M. J. ASH-
1. Oligomer
of Cryoinjury Dissociation
Me&an&m. W. N. FISHBEIN Branch, Armed Forces Institute Washington, DC 20305).
and Cryoas the General
(Biochemistry of Pathology,
ANNUAL
MEETING
Since cryoinjury and cryophylaxis have both been documented in pure macromolecular systems, a general mechanism must be formulated at the molecular level and then extrapolated to cells and tissues ; certainly one cannot extrapolate mechanical or membrane damage theories to the molecular level. An earlier interpretation [Crl/obiology 6, 261, (1969)] is here extended to include the effect of cryoprotectants. The primary lesion is presumed to be the dissociation of oligomeric macromolecules during freezing, following, perhaps, by partial denaturation (peptide backbone unfolding). On thawing, biologic damage develops if protomer reassociation is imperfect due to (I) failure to reassociate. (2) incorrect association with homologous protomers, or (3) association with heterologous protomers. Dissociation of certain entropic polymers upon sufficient fall in temperature is the basic mechanism, aided by increasing ionic strength, which “salts apart” electrostatic and H-bonds. Cryoprotectants. which are primarily shielded dipoles, interact directly with the macromolecules to insulate these bonds from the action of salts. Inevitably, at high enough concentrations, cryoprotective agents will themselves dissociate oligomers, by insulating electrostatic charges, forming nontransmitted Hbonds, and favoring exposure of hydrophobic residues. Permeant and impermeant agents function similarly, their effect,iveness being related. not to their molar concentration, but to that, of their dipole sites. Since colligative properties are not essential to the action of pcrmeant agents, and since impcrmeant agents can only protect the external plasma membrane. which is unlikely to be the sole site of damage in cells other than the erythrocyte, the logical approach is to study penetrating agents at low concentrations. Some evidence for this view will be presented in other communications (Abstracts 20,44. 61). 17. Fluorescence Detection of Cryoprotectant Interactions. M. J. RUWART.* J. F. HOLLAKD*$,
A. HAUG*~ (*Department of Biophysics and MSU/AEC Plant Research Laboratory; IDepartment of Biochemistry, Michigan State University, E. Lansing. MI 48823).
AKD
Alt,hough the ability of certain compounds to protect living cells against freeze-thaw damage has been well-documented, little is known about their interaction with other cryoprotectants, biological macromolecules, and cell organclles at the molecular level. A computer-centered spectrofluorimeter has been developed which simultaneously records absorbance and fluorescence spectra. and calculates a new parameter, the partial quantum efficiency. When tryptophan was used as a probe fluorophore,
311
ABSTRACTS
its partial quantum efficiency indicated the presence of weak molecular interactions between dimethyl sulfoxide and dextran (MW = 86,000). Such interactions were not observed in other cryoprotectant combinations tested (e.g., glucose and dimet.hyl sulfoxide). These findings may in part explain the results of Ilarow et al. [Arch. Surg. 91, 572 (1965)] who reported that the dextramdimethyl sulfoxide combination did not protect frozen rat hearts, e\-en though both compounds enhanced survival when used separately. Moreover, a model of slight protcin expansion may be employed to interpret the results obtained upon the addition of low molecular weight crgoprotectants to bovine serum albumin. In contrast, high molecular weight compounds appear to constrict its structurc. (Supported in part by Contra1 AT(ll-l)-1338 from the U. S. Atomic Energy Commission and Grant GM-0142246 from t,he National Institutes of Health, U. S. Public Health Service.) 18. The Protective Action of Vuriotls Polymers, Including Polyvilzylpyrrolidolze, Dextran and Hydroxyethyl to Mammalian
Starch Against Freezing Damage Cells in Tissue Culture. M. J.
ASHWOOD-SMITH, C. WARBY,* W. CONNOR” (Department of
University
G. BECKER,* AND Biological Sciences, of Victoria, Victoria. B. C., Canada).
Chinese hamster cells in tissue culture were assayed for viability using a Puck Type Survival test after freezing and thawing in the presence of added polymers with a variety of cooling and thawing rates. The PVP samples of low molecular weight were ineffective. The PVP samples of molecular weight of 40.000 daltons (average) or greater gave survival values in the region of 50% (DMSOtreated cells frozen under identical conditions gave values in the region of 70%). Hydroxyethyl starch (HES) was almost as good as the best PVP samples but a number of dextran samples (molecular weight varying from 10,000 daltons up to and including 500,000 daltons) were totally without protective action over the range of cooling rates (5”C/ min to 3OO”C/min) used in these experiments. The total lack of action with dextran is most interesting and these results will be discussed in relation to a new theory of polymer protect.ion. 19. The Cryoprotective Effect of Certain Zwitterionic Buffers. D. M. ROBINSON (American Red Cross Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, MD 20014). With the advent of a series of new, nonvolatile, hydrogen ion buffers, having pK, values within the range 6-S [Good et al., Biochemie 5, 467 (1966)] many workers using mammalian cell cultures have
ANNUAL
MEETING
Groups of rats of both sexes were exposed to temperatures of 1°C and -10°C for 60 min prior to orogastric intubation, whereby 2 &i/l00 g body wt was administered. 4Ca level in bone was measured by sbandard monitoring equipment 24 hr after intubation. Exposure to 1°C did not produce significant difference in radiocalcium bone uptake (63 nmCi/g bone wt), when compared to the controls (57 nmCi/g bone wt). In rats exposed to -10°C the mean bone uptake was 92 nmCi/g bone wt, an increase of 61%. A 1-hr exposure to -10°C after intubation also produced an increase (83%) in “Ca uptake (mean 105 nmCi/g bone wt). These results suggest that exposure to low temperature, both prior and after 45Ca administration produces significant increase in calcium bone uptake. Several possible mechanisms should be considered, including hormonal stimulation by hypothermia or increased oxygen consumption and ATP synthesis. 13. Quantitative
Changes in Total Nucleic and Proteins of the Isolated Rabbit During Normothermic and Hypothermic
Acids Heart Per-
fusion. G. I. MALININ, R. M. NARDONE. .4x11 J. (Department of Biology, GeorgeA. PA~SKA
SESSION
ABSTRACTS town University, Washington, DC 20007 and Department of Biology, Catholic University of America, Washington, DC 20011).
Thirty-one rabbit hearts were perfused for 26 hr at normothermic and hypothermic temperatures utilizing a Lindbergh-Rockefeller Institute pulsatile perfusion apparatus. The perfusate consisted of Hanks’ balanced salt solution supplemented with 10% (v/v) rabbit serum. Each atrium and ventricle was then analyzed for total DNA, RNA and protein content utilizing a modified Schmidt-Thannhauser procedure. As compared with controls. levels of DNA and protein were reduced significantly (P < 0.05) in the atria and vent,ricles of the perfused hearts while RNA levels remained unchanged with the exception of the right ventricle where they also depressed. The RNA/DNA ratios, however, were significantly elevated (P < 0.05) in ventricles but remained within normal ranges in atria. The DNA/ protein ratios were depressed in the right ventricles and remained unchanged in the rest of the heart. The RNA/protein ratios were higher in all cardiac chambers. (Supported by Contract N0014-69-A0220 from the Office of Naval Research.)
B : MOLECULAR
14. Fish Blood Glycoproteins with Antifreeze Properties. J. A. RAYMOSD,* AND A. L. DEVRIES
(Physiological Research Laboratory, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92037). The blood of some species of Antarctic fish has been shown to contain freezing point depressing glycoproteins. In a solution having normal colligative properties, the freezing point depression is solely a function of the number of particles in solution. Unlike other solutes in the blood, the glycoproteins are large molecules (2000-20,000 daltons) and have an effect on the freezing point depression two orders of magnitude greater than would be expected on a purely molar basis. This paper is concerned with some of the other ways in which this nonclassical freezing behavior is manifested. Evidence that the glycoproteins are incorporated into the ice phase in a freezing solution is presented. Supercooling of the blood in the presence of an ice seed has also been observed. A possible means by which these phenomena may occur is suggested. (Supported by Grant No. GV 27327 from the National Science Foundation.)
MECHANISMS
WOOD-SMITH, AKD C. WARBY* (Department of Biological Sciences, University of Victoria, Victoria: B.C., Canada).
Experiments are reported that clearly demonstrate, in vitro, the protective action of a number of cryoprotective agents against the inactivat.ion of the enzyme catalase by freezing and thawing. The most significant outcome of these investigations is the very large difference in effectiveness between PVP and either glycerol or dimethyl sulfoxide. The amounts required, on a molar basis, to protect enzyme activity by 50% were approximately 2 X IOF2M (glycerol or DMSO) and 1 X IO-’ III (PVP of av MW 40,000 daltons). This very large difference in effectiveness, nearly 1000 even on a weight basis, is difficult to explain in terms of the slight but definite colligative properties of PVP. Other polymers such as dextran protect catalase but are far less effective than PVP. Possible mechanisms of PVP protection will be discussed. (Supported by Canadian Medical Research Council Grant MA4055.) 16. Organochemistry phylaxis
15. Protective Efiects of Low an,d High Molecular Weight Compounds on the Stability of Catalase Subjected to Freezing and Thawing. M. J. ASH-
1. Oligomer
of Cryoinjury Dissociation
Me&an&m. W. N. FISHBEIN Branch, Armed Forces Institute Washington, DC 20305).
and Cryoas the General
(Biochemistry of Pathology,
ANNUAL
MEETING
Since cryoinjury and cryophylaxis have both been documented in pure macromolecular systems, a general mechanism must be formulated at the molecular level and then extrapolated to cells and tissues ; certainly one cannot extrapolate mechanical or membrane damage theories to the molecular level. An earlier interpretation [Crl/obiology 6, 261, (1969)] is here extended to include the effect of cryoprotectants. The primary lesion is presumed to be the dissociation of oligomeric macromolecules during freezing, following, perhaps, by partial denaturation (peptide backbone unfolding). On thawing, biologic damage develops if protomer reassociation is imperfect due to (I) failure to reassociate. (2) incorrect association with homologous protomers, or (3) association with heterologous protomers. Dissociation of certain entropic polymers upon sufficient fall in temperature is the basic mechanism, aided by increasing ionic strength, which “salts apart” electrostatic and H-bonds. Cryoprotectants. which are primarily shielded dipoles, interact directly with the macromolecules to insulate these bonds from the action of salts. Inevitably, at high enough concentrations, cryoprotective agents will themselves dissociate oligomers, by insulating electrostatic charges, forming nontransmitted Hbonds, and favoring exposure of hydrophobic residues. Permeant and impermeant agents function similarly, their effect,iveness being related. not to their molar concentration, but to that, of their dipole sites. Since colligative properties are not essential to the action of pcrmeant agents, and since impcrmeant agents can only protect the external plasma membrane. which is unlikely to be the sole site of damage in cells other than the erythrocyte, the logical approach is to study penetrating agents at low concentrations. Some evidence for this view will be presented in other communications (Abstracts 20,44. 61). 17. Fluorescence Detection of Cryoprotectant Interactions. M. J. RUWART.* J. F. HOLLAKD*$,
A. HAUG*~ (*Department of Biophysics and MSU/AEC Plant Research Laboratory; IDepartment of Biochemistry, Michigan State University, E. Lansing. MI 48823).
AKD
Alt,hough the ability of certain compounds to protect living cells against freeze-thaw damage has been well-documented, little is known about their interaction with other cryoprotectants, biological macromolecules, and cell organclles at the molecular level. A computer-centered spectrofluorimeter has been developed which simultaneously records absorbance and fluorescence spectra. and calculates a new parameter, the partial quantum efficiency. When tryptophan was used as a probe fluorophore,
311
ABSTRACTS
its partial quantum efficiency indicated the presence of weak molecular interactions between dimethyl sulfoxide and dextran (MW = 86,000). Such interactions were not observed in other cryoprotectant combinations tested (e.g., glucose and dimet.hyl sulfoxide). These findings may in part explain the results of Ilarow et al. [Arch. Surg. 91, 572 (1965)] who reported that the dextramdimethyl sulfoxide combination did not protect frozen rat hearts, e\-en though both compounds enhanced survival when used separately. Moreover, a model of slight protcin expansion may be employed to interpret the results obtained upon the addition of low molecular weight crgoprotectants to bovine serum albumin. In contrast, high molecular weight compounds appear to constrict its structurc. (Supported in part by Contra1 AT(ll-l)-1338 from the U. S. Atomic Energy Commission and Grant GM-0142246 from t,he National Institutes of Health, U. S. Public Health Service.) 18. The Protective Action of Vuriotls Polymers, Including Polyvilzylpyrrolidolze, Dextran and Hydroxyethyl to Mammalian
Starch Against Freezing Damage Cells in Tissue Culture. M. J.
ASHWOOD-SMITH, C. WARBY,* W. CONNOR” (Department of
University
G. BECKER,* AND Biological Sciences, of Victoria, Victoria. B. C., Canada).
Chinese hamster cells in tissue culture were assayed for viability using a Puck Type Survival test after freezing and thawing in the presence of added polymers with a variety of cooling and thawing rates. The PVP samples of low molecular weight were ineffective. The PVP samples of molecular weight of 40.000 daltons (average) or greater gave survival values in the region of 50% (DMSOtreated cells frozen under identical conditions gave values in the region of 70%). Hydroxyethyl starch (HES) was almost as good as the best PVP samples but a number of dextran samples (molecular weight varying from 10,000 daltons up to and including 500,000 daltons) were totally without protective action over the range of cooling rates (5”C/ min to 3OO”C/min) used in these experiments. The total lack of action with dextran is most interesting and these results will be discussed in relation to a new theory of polymer protect.ion. 19. The Cryoprotective Effect of Certain Zwitterionic Buffers. D. M. ROBINSON (American Red Cross Blood Research Laboratory, 9312 Old Georgetown Road, Bethesda, MD 20014). With the advent of a series of new, nonvolatile, hydrogen ion buffers, having pK, values within the range 6-S [Good et al., Biochemie 5, 467 (1966)] many workers using mammalian cell cultures have